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Collagen Type I and Decorin Expression in Tenocytes Depend On Wagenhäuser et al. BMC Musculoskeletal Disorders 2012, 13:140 http://www.biomedcentral.com/1471-2474/13/140 RESEARCH ARTICLE Open Access Collagen type I and decorin expression in tenocytes depend on the cell isolation method Markus U Wagenhäuser1†, Matthias F Pietschmann1*†, Birte Sievers1, Denitsa Docheva2, Matthias Schieker2, Volkmar Jansson1 and Peter E Müller1 Abstract Backround: The treatment of rotator cuff tears is still challenging. Tendon tissue engineering (TTE) might be an alternative in future. Tenocytes seem to be the most suitable cell type as they are easy to obtain and no differentiation in vitro is necessary. The aim of this study was to examine, if the long head of the biceps tendon (LHB) can deliver viable tenocytes for TTE. In this context, different isolation methods, such as enzymatic digestion (ED) and cell migration (CM), are investigated on differences in gene expression and cell morphology. Methods: Samples of the LHB were obtained from patients, who underwent surgery for primary shoulder arthroplasty. Using ED as isolation method, 0.2% collagenase I solution was used. Using CM as isolation method, small pieces of minced tendon were put into petri-dishes. After cell cultivation, RT-PCR was performed for collagen type I, collagen type III, decorin, tenascin-C, fibronectin, Scleraxis, tenomodulin, osteopontin and agreccan. Results: The total number of isolated cells, in relation to 1 g of native tissue, was 14 times higher using ED. The time interval for cell isolation was about 17 hours using ED and approximately 50 days using CM. Cell morphology in vitro was similar for both isolation techniques. Higher expression of collagen type I could be observed in tenocyte-like cell cultures (TLCC) using ED as isolation method (p < 0.05), however decorin expression was higher in TLCC using CM as isolation method (p < 0.05). Dedifferentiation potential seemed to be similar for both isolation techniques. Conclusion: In summary tenocyte-like cells can be obtained with both isolation methods (ED and CM) from the LHB. As no obvious disadvantage could be seen using ED, this method is more suitable for clinical use, as time for cell isolation is shorter and a remarkably higher number of cells can be obtained. However, both isolation methods can further be improved. Keywords: Tenocytes, Tissue engineering, Isolation method, Gene expression Backround procedures. In Europe, allogenic tendons grafts are not Tendon and ligament injuries are common injuries in used routinely [2]. Moreover, in 2006 Moore et al. ad- Orthopedics. The most important injuries are ruptures vised not to use allogenic tendon grafts for rotator cuff of the anterior cruciate ligament in the knee, rotator cuff reconstruction, because the clinical outcome was com- tears in the shoulder and ruptures of the Achilles ten- parable to simple debridement and had an increased risk don [1]. of infection and host-versus-graft-reaction [3]. Up to date, the treatment of severe rotator cuff tears In future, an alternative approach for irreparable rota- remains challenging. The dimension of these ruptures tor cuff tears might be tendon tissue engineering (TTE). often does not allow a complete reconstruction. Only It tries to create tendon tissue of good quality in vitro specific groups of patients benefit from tendon transfer aiming to take over its specific function in situ after implantation. An important issue for the TTE is to find * Correspondence: [email protected] out which cell type is the most effective for in vitro cell †Equal contributors 1Department of Orthopaedic Surgery, Ludwig-Maximilians-University Munich - seeding. The ideal cell type should fulfill certain cri- Campus Grosshadern, Marchioninistr 15, 81377, Munich, Germany teria. Firstly, it should be possible to isolate cells from Full list of author information is available at the end of the article © 2012 Wagenhäuser et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Wagenhäuser et al. BMC Musculoskeletal Disorders 2012, 13:140 Page 2 of 8 http://www.biomedcentral.com/1471-2474/13/140 autologous tissue to avoid host-versus-graft reaction af- was performed by the senior author (P.E.M), an experi- ter implantation. Secondly, an adequate number of vital enced shoulder surgeon. The study was carried out fol- cells should be available after the cell isolation. Thirdly, lowing the regulations of the Medical Center Ethics these cells should have the ability to expand in vitro as Committee of the Ludwig-Maximilians-University of well as to maintain phenotypic stability throughout the Munich (ethical grant number: 063–09). passages [4]. Altogether, seven tissue samples of the LHB were col- So far, fibroblasts, mesenchymal stem cells and teno- lected from patients with an average age of 60 years cytes have been investigated [5-7]. Tenocytes might be (± 8.9 years). Tendon samples were carried to the la- the most suitable cells as no differentiation during boratory in cell culture medium (DMEM/HAM’s F12, in vitro cultivation is necessary. Tenocytes have been Biochrom, Berlin, Germany) where cell isolation was im- shown to grow in vitro without signs of senescence [8]. mediately performed. Under sterile conditions tendons There is also evidence that cell proliferation of tenocytes were washed three times with PBS buffer (Biochrom, in vitro is comparable to mesenchymal stem cells [9]. Berlin, Germany) and cut into small pieces. For the fol- In this context, the long head of the biceps tendon lowing cell isolation previously published methods were (LHB) seems to be promising for tenocyte isolation. A used [6,16,17]. Briefly, the sheath and surrounding para- (partial or complete) rupture of the LHB is often found tenon were removed and the tendons were minced into in association with rotator cuff tears [10,11]. For this small pieces. At this stage, slices from each sample were reason tenotomy of the LHB is performed regularly dur- randomly assigned to perform the following isolation ing rotator cuff reconstruction [12-14]. Donor side mor- procedure. bidity after tenotomy of the LHB is negligible and patient satisfaction is high [15]. ED method There are two different methods to isolate tenocytes One part of the tendon slices was incubated in 0.2% col- from tendon tissue. Tenocytes can be isolated using en- lagenase I solution (Sigma-Aldrich, Steinheim, Germany) zymatic digestion (ED) of the extracellular matrix com- for approximately 18 hours in 37°C, CO2 5%. After di- pounds [6,16]. Another alternative is to obtained tenocytes gestion, cells were filtered (100 μm) (BD Biosciences, by cell migration (CM) as cells migrate out of the tissue Erembodegem, Belgium), the suspension was washed explants after culturing in cell culture medium [17]. three times with PBS buffer and centrifuged (Heraeus, Tenocytes synthesize specific proteins of the extracellu- Hanau, Germany) at 300 g for 5 minutes. Before cell cul- lar matrix, which has a highly ordered composition [18]. tivation, tenocytes were counted using a heamocytometer Collagen type I, collagen type III, decorin, tenascin-C are and trypan blue staining to distinguish between dead and fundamental proteins in the extracellular matrix of ten- vital cells. dons. High expression levels of collagen type I and decorin seem to be essential for cells to be suitable for TTE appli- CM method cations, as they play an important role in tissue forma- The other part of the tendon slices were placed into tion [19,20]. Scleraxis and tenomodulin are commonly petri-dishes filled with 10 ml cell culture medium used as markers for tenogenic differentiation [21-23]. (DMEM/HAM’s F12), supplemented with 10% FCS, and Tenascin-C and fibronectin are two glycoproteins of were subcultivated (37°C, CO2 5%). Culture medium was the extracellular matrix, which are essential for cell-cell changed every second day. After a few days, the first col- and cell-matrix interactions [24]. Aggrecan and osteo- onies of migrating tenocytes around the slices could be pontin are located predominantly in the extracellular seen. After approximately 3 weeks, the tendon slices were matrix of cartilage and bone. carefully transferred into new petri-dishes. Altogether The aim of this study was to examine if the LHB is three migration cycles were performed. Before cell culti- suitable as cell source for TTE. Additionally, we com- vation the total number of cells was counted using a pared two different isolation methods, ED and CM. In haemocytometer and trypan blue. order to investigate the influence of these isolation meth- ods on tenocyte-like cell cultures (TLCC), we analyzed Tendon cell cultures cell morphology and gene expression. We hypothesized The isolated tenocytes were placed in culture flask in that both isolation methods deliver similar cell yield and DMEM/HAM`s F12 (1:1) supplemented with 10% FCS, show no differences in gene expression. 2 mM L-Glutamin, ascorbic acid 1250 μg/ml, amino acids, Penicillin/Streptomycin 60 μg/ml and Amphoteri- Methods cin B 25 ng/ml (Biochrom, Berlin, Germany). The seed- Cell isolation procedure ing density for each isolation method was as follows: Tendon samples were obtained from patients, who under- CM-714 cells/cm2 and ED-2857 cells/cm2. As soon as went surgery for a primary shoulder arthroplasty. Surgery the cultured cells reached 80-90% confluence, they were Wagenhäuser et al. BMC Musculoskeletal Disorders 2012, 13:140 Page 3 of 8 http://www.biomedcentral.com/1471-2474/13/140 treated with 0.05% trypsin/0.02% ethylenediaminetetra- minutes at 25°C, following incubation at 42°C for 60 aciticacid (EDTA) (Biochrom, Berlin, Germany) and sub- minutes. cDNA samples were stored at −20°C until fur- cultured. Osteoblasts (HOB lot: 540X130406) and ther use.
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